Septicemia is the thirteenth leading cause of death in humans, accounting for up to 100,000 deaths and $5 to $10 billion of health care expenditures annually in the United States, alone (Increase in National Hospital Discharge Survey rates for septicemia—United States, 1979-1987 MMWR 1990, 39:31-34; Skelter et al. Arch. Microbiol. 1995, 164:383-389). It is the most common cause of death in medical and surgical intensive care units, and is associated with a mortality rate of 40 to 90%.
Septicemia is caused by the release of endotoxin, also referred to herein as lipopolysaccharide (LPS), from the outer wall of gram negative bacteria into the blood stream. LPS or endotoxin is released from the outer membrane of the bacteria when they multiply, die, or lyse (Rietschel et al. FASEB J. 1994, 8:217-225). Following its release, this toxin produces a cascade of complex events which ultimately results in organ failure, irreversible shock and death. By binding to cell membrane proteins or specific cell membrane receptors, LPS acts on a number of different cell types, including endothelial cells, neutrophils, monocytes and macrophages to induce the release of a number of mediators, including oxygen free radicals, nitric oxide, metabolites of arachidonic acid, thromboxane, prostacyclin, and platelet activating factor, chemoattractants, interleukin (IL)-8 and leukotriene B4, cytokines, IL-1, tumor necrosis factor (TNF-α), and proteases which are important in the pathophysiology of endotoxin induced organ injury and septic shock (Akarasereenont et al. Eur. J. Pharmacol. 1995, 273:121-128; Brigham et al. Am. Rev. Respir. Dis. 1986, 133:913-927; Morrison Ann. Rev. Med. 1987, 38:417-432; Williams et al. Surgery 1992, 112:270-277; Wright Current Opinion in Immunol. 1991, 83-91).
Despite the prevalence and severity of this disease, rapid diagnosis at an early stage is still difficult. The diagnosis of septicemia is currently based upon on clinical signs and symptoms, including clinically significant hypotension (systolic blood pressure ≦90 mmHg) with suspected presence of infection, fever or hypothermia, tachycardia, tachypnea, lactic acidosis, white blood cell count >12,000 or <4,000, with or without positive blood cultures. However, by the time such clinical signs and symptoms manifest, irreversible organ and tissue damage may have already occurred. Further, results from blood cultures require at least 24 hours and do not always provide a definitive diagnosis since only 35% of patients with septicemia have positive blood cultures.
The only tests currently available for detection of endotoxin in biological fluids, or pharmacological and industrial solutions and products, are based on the availability of lysates of amebocytes isolated from the hemolymph of Limulus and Tachypleus (rare horseshoe crabs). The Limulus amebocyte lysate (LAL) test is based on the ability of endotoxin to coagulate with amebocyte lysate. These amebocytes have a coagulation system believed to be a prototype of mammalian blood coagulation that involves the sequential activation of proenzymes (Zhang et al. J. Clin. Microbiol 1994 32:416-422). Endotoxin activates the initial enzyme (factor C) of the LAL coagulation system leading to conversion of coagulogen, a clottable protein, into coagulin and peptide C. Visible formation of a gel clot indicates endotoxin activation of LAL and serves as the basis for the gel-clot method for detection of endotoxin. However, this particular method is not quantitative. In order to make this assay quantitative, a turbidimetric kinetic assay which uses a toxinometer was developed (Kambayashi et al. J. Biochem. Biophys. Methods 1991, 22:93-100). In addition, a chromogenic substrate for the clotting enzyme was developed (Thomas et al. Clinica Chimica Acta. 1981, 116:63-68). The addition of this chromogenic substrate to the assay increased the sensitivity of the LAL assay 10 to 100 times greater than that of the gel-clot method. A spectrophotometric, calorimetric (toxicolor) assay was developed by addition of a dye, N-(-1 naphthyl)-ethylene-diamine, to the chromogenic substrate assay, (Minobe et al. Eur. J. Clin. Chem. Clin. Biochem. 1991, 32:797-803).
However, the LAL test is not sufficiently specific enough for clinical diagnosis of septicemia in human plasma. LAL tests are affected by β-glucans, β-glucan-mycotic containing reactive products, and rinses from cellulose-based dialyzers (Morita et al. FEBS Letters 1981 129:318-321). Further, substances in human blood having nonspecific amidolytic activities, such as factor Xa, thrombin, and trypsin act directly on the chromogenic substrate (Obayashi J. Lab. Clin. Med. 1984, 104:321-330) and produce false positive results. Inhibitors such as α2-plasmin inhibitor, antithrombin III, and α1-antitrypsin produce false negative results (Obayashi et al. Clinica Chimica Acta 1985, 149:55-65). Moreover, the LAL test is inhibited or enhanced by many substances including antibiotics, hormones, heavy metals, amino acids, alkaloids, carbohydrates, plasma proteins, enzymes, and electrolytes in the sample solution (Pfeiffer, M. and Weiss, A. R, “Removal of Lal-test interfering low molecular weight substances by ultrafiltration”, Detection of Bacterial Endotoxin with the Limulus Amebocyte Lysate Test, W W Levin and J T Novitsky (eds), Alan R. Liss, Inc., New York, N.Y., 1987, pp. 251-262). To remove such interfering substances, methods for treatment of plasma samples, with heating and dilution, perchloric acid, chloroform, ether, acid, alkali, detergents, or ultrafiltration have been tested (Pfeiffer, M. and Weiss, A. R. supra; Obayashi, T. J. Lab. Clin. Med. 1984, 104:321-330). However, dilution of the sample results in dilution of the endotoxin level and reduces the sensitivity of detecting low levels of endotoxin in the sample. Attempts at ultrafiltration to remove high molecular weight interfering substances have also proved unsuccessful because the pore size is too small and endotoxin may be adsorbed on the ultrafiltration membrane (Nawata et al. J. Chromatography 1992, 597:415-424).
Minobe et al. developed a method for eliminating interfering substances in the sample solution by adsorbing endotoxin onto immobilized histidine subsequently assayed with the LAL chromogenic substrate and toxinometer or toxicolor test (Minobe et al. Eur. J. Clin. Chem. Clin. Biochem. 1994, 32:797-803). However, binding of endotoxin to immobilized histidine can be affected by globulins and transferrin in human plasma which bind to endotoxin. Further, the histidine mobilization method is dependent upon the reaction time and dilution of the sample to increase the sensitivity of the measurement.
LAL enzyme-linked immunosorbent assays (ELISA) for the detection of endotoxin have also been developed with coagulogen and Limulus peptide C (Zhang et al. J. Clin. Microbiol. 1994, 32:416-422). However, these ELISAs are also affected by substances in human blood which activate or inhibit LAL as described above and like other LAL assays depend on the availability of the rare horseshoe crabs which are diminishing in populations.
Accordingly, there exists a need for a quick and reliable assay for the determination of endotoxin in a sample.